Separation Of Cells Based On Size And Affinity Using Paper Microfluidic Device

ABSTRACT

A microfluidic device includes a first layer of a porous material with pores having a first average pore size and a liquid-receiving area through which a liquid sample is received into the microfluidic device. A second layer of another porous material, with pores of a second average pore size, is stacked below the first layer and has a channel with a starting end positioned at least in part in an overlapping manner with the liquid-receiving area. The channel has a terminating end extending laterally at a predetermined wicking distance from the starting end. The first average pore size and the second average pore size cause a wicking effect in which at least some of the liquid sample flows along the channel at least a portion of the wicking distance between the starting end and the terminating end.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage bypass continuation-in-part ofInternational Application No. PCT/US2017/013065, filed on Jan. 11, 2017,and titled “Separation Of Cells Based On Size And Affinity Using PaperMicrofluidic Device,” which claims priority to and benefit of U.S.Provisional Patent Application Ser. No. 62/277,810, filed on Jan. 12,2016, and titled “Separation Of Cells Based On Size And Affinity UsingPaper Microfluidic Device,” each of which is hereby incorporated byreference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to paper-based analytical andbioanalytical sensors for separation, detection, and quantification ofcells from complex samples.

BACKGROUND OF THE INVENTION

Paper-based microfluidic devices have emerged as a platform that iscapable of supporting the development of a number of useful analyticaland bioanalytical sensors, the capabilities of which range from thedetection of environmental contaminants to metabolites in blood plasma.Paper-based sensors often produce colorimetric results, which allow datato be interpreted rapidly at the point-of-use in a manner that is eitherqualitative (i.e., by eye) or quantitative through the use of simplereaders. Utilizing paper as a substrate to develop analytical assays isbeneficial because the infrastructure required to produce the analyticalassays is minimal (e.g., a printer, heating element, and pipette), rawmaterials are inexpensive and ubiquitous (cents per sheet), and devicescan be prototyped rapidly (within minutes from conception to use). Bypatterning paper with hydrophobic barriers, hydrophilic channels can bedesigned to control the wicking of fluids by capillary action. Complex,three-dimensional microfluidic networks can be fabricated from eitherstacking multiple layers of paper or folding a single layer of paper(i.e., origami). Simple design rules provide access to many differentarchitectures of fluidic networks, which can facilitate the manufactureof devices that range from one-step, field-deployable diagnostic toolsto sophisticated paper “machines.”

With the considerable interest in this field of research, a glaringoversight has been the lack of applications of paper-based microfluidicdevices for the separation or detection of cells. This omission appearsto be caused based on a perspective in which paper is viewed as apassive substrate, instead of being viewed as a component that isfundamental to the function of the microfluidic device. Consequently,paper has been applied only to the filtration of all cells from plasmaor to the separation of misformed (i.e., sickled) red blood cells, or asa scaffold for the study of cultures of mammalian cells. However, theability to detect cells has significant value in applications relatedto, among others, personalized healthcare, monitoring of livestock, anddetermining the quality of food and water. These important capabilitiesare currently only available in established economies with centralizedlaboratories that are equipped with modern instrumentation and thatinclude an educated workforce. Consequently, a significant percentage ofthe world's population—particularly those living in low-income andmiddle-income countries—have limited access to tools that coulddrastically improve the quality of life. Accordingly, paper has thepotential to enable new classes of biological separations, analyticalsensors, and point-of-use assays for underrepresented populations acrossthe globe.

SUMMARY OF THE INVENTION

According to one aspect of the present disclosure, a three-dimensional(“3D”) microfluidic device is capable of supporting the development ofanalytical and bioanalytical sensors. The device allows for separationand/or quantification of the cells in whole human blood viasize-exclusion determined by pore size and via affinity separation bybiochemical functionalization of a porous material, such as paper, asdescribed below in reference to the drawings. By way of example, thepotential of the microfluidic device is directed to at least a futurenew low-cost platform for the identification of a critical hematologicalindex (hematocrit), i.e., the ratio of packed red blood cell volume tototal blood volume. Additional potential applications includepersonalized healthcare, monitoring of livestock, and determining thequality of food and water.

According to one aspect of the present disclosure, a microfluidicdevice, includes a first layer of a porous material with pores having afirst average pore size, the first layer having a liquid-receiving areathrough which a liquid sample is received into the microfluidic device.A second layer of another porous material, which is the same ordifferent than the porous material of the first layer, is stacked belowthe first layer, the second layer having pores of a second average poresize. A channel is positioned within the second layer and has a startingend positioned at least in part in an overlapping manner with theliquid-receiving area. The channel has a terminating end extendinglaterally at a predetermined wicking distance from the starting end. Thefirst average pore size and the second average pore size cause a wickingeffect in which at least some of the liquid sample flows along thechannel at least a portion of the wicking distance between the startingend and the terminating end.

According to another aspect of the present disclosure, a method isdirected to providing a microfluidic device and includes providing afirst layer of a porous material with pores having a first average poresize, the first layer having a liquid-receiving area through which aliquid sample is received into the microfluidic device. The methodfurther includes stacking below the first layer a second layer of thesame or different porous material having pores of a second average poresize, and positioning a channel within the second layer. The channel hasa starting end positioned at least in part in an overlapping manner withthe liquid-receiving area of the first layer, the channel having aterminating end extending laterally at a predetermined wicking distancefrom the starting end. The first average pore size and the secondaverage pore size are selected such that, upon the receiving of theliquid sample, a wicking effect is caused in which at least some of theliquid sample flows along the channel at least a portion of the wickingdistance between the starting end and the terminating end.

According to yet another aspect of the present disclosure, microfluidicdevice includes a sample-addition layer of a first porous material withpores having a first average pore size. The sample-addition layer has aliquid-receiving area through which a liquid sample is received into themicrofluidic device. The microfluidic device further includes asample-splitting layer located adjacent to the sample-addition layer,the sample-splitting layer having a first aperture for receiving a firstportion of the liquid sample and a second aperture for receiving asecond portion of the liquid sample. The microfluidic device alsoincludes a separation membrane located adjacent to the first aperture ofthe sample-splitting layer, the separation membrane receiving only theportion of the liquid sample from the first aperture. The microfluidicdevice also includes a readout layer of a second porous material locatedadjacent the separation membrane, the second porous material havingpores with a second average pore size that is different than the firstaverage pore size. The readout layer has a first channel configured toreceive in a starting end the first portion of the liquid sample fromthe sample-splitting layer, via the separation membrane. The firstchannel has a terminating end extending laterally at a predeterminedwicking distance from the starting end, the first portion of the liquidsample flowing at least in part along the wicking distance to indicate afirst value of the liquid sample. The readout layer further has a secondchannel positioned adjacent to the first channel for receiving thesecond portion of the liquid sample, the second channel including astored reagent that reacts with the second portion of the liquid sampleto indicate a second value of the liquid sample.

Additional aspects of the present invention will be apparent to those ofordinary skill in the art in view of the detailed description of variousembodiments, which is made with reference to the drawings, a briefdescription of which is provided below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is an illustration showing selection of paper pore sizes forpreventing migration of cells.

FIG. 1B is an illustration showing a broad range of cell sizes.

FIG. 1C is an illustration showing selection of paper pore sizes forpermitting migration of cells.

FIG. 2A is an illustration of a disassembled 2-layer microfluidicdevice.

FIG. 2B is an illustration of the microfluidic device of FIG. 2A shownin assembled form and indicating different hematocrits.

FIG. 3A is an illustration showing the effect of paper pore size on theperformance of a plurality of paper-based hematocrit assays.

FIG. 3B is an illustration showing the effect of a threshold pore sizeon the performance of a paper-based hematocrit assay.

FIG. 4A is an illustration showing separation of large granulocytes fromsmall lymphocytes.

FIG. 4B is an illustration showing separation of somatic cells frombacteria.

FIG. 5 is an illustrating showing separation of cells in paper based onpartitioning, with erythrocytes (but not leukocytes) binding to paperthat has been grafted with dextran.

FIG. 6A is an illustration showing an integrated paper-basedmicrofluidic device for separating multiple biological species from asingle sample.

FIG. 6B is an illustration showing another integrated paper-basedmicrofluidic device having multiple adhesive areas per layer forseparating multiple biological species.

FIG. 7 is a graphical representation of various hematocrit percentagesfor samples of whole blood using microfluidic devices treated withsodium chloride and ethylenediaminetetraacetic acid (EDTA).

FIG. 8 is a graphical representation of various hematocrit percentagesfor samples of whole blood using microfluidic devices treated with EDTAon two different sections of the microfluidic devices.

FIG. 9 is a graphical representation of various hematocrit percentagesfor samples of isolated red blood cells in Alsever's solution.

FIG. 10 is a graphical representation of various hematocrit percentagesfor samples of whole blood using microfluidic devices treated withsodium chloride with and without EDTA.

FIG. 11 is a graphical representation comparing differences inperformance between isolated red blood cells (“RBCs”) and whole blood.

FIG. 12 is a graphical representation illustrating a comparison ofperformance of isolated RBCs on microfluidic devices that are treatedand are not treated with sodium chloride.

FIG. 13A is a perspective view illustration of a disassembledmulti-layer microfluidic device.

FIG. 13B is top view illustration of the microfluidic device of FIG. 13Ashown in assembled form.

FIG. 13C is back view illustration of the microfluidic device of FIG.13A shown in assembled form.

FIG. 14A illustrates the microfluidic device of FIG. 13A with a firstvisual readout of a hematocrit (“Hct”) and hemoglobin (“Hb”) amounts.

FIG. 14B illustrates the microfluidic device of FIG. 13A with a secondvisual readout of the Hct and Hb amounts.

FIG. 14C illustrates the microfluidic device of FIG. 13A with a thirdvisual readout of the Hct and Hb amounts.

FIG. 15A is an illustration of a disassembled multilayer microfluidicdevice.

FIG. 15B illustrates the relationship between blood transport distanceand hematocrit range in the multilayer microfluidic device of FIG. 15Aand using an original device design with 50 microliters (“μL”) of blood.

FIG. 15C illustrates data showing correlation between hematocritpercentage and normalized distance for original device designs using 50μL of blood, as illustrated in FIG. 15B, and scaled device designs usingonly 10 μL of blood, as illustrated in FIG. 15D.

FIG. 15D illustrates a scaled device using only 10 μL of blood.

FIG. 16A is a schematic of a paper-based assay in which differentialtransport distances across multiple grades of paper are used to bincorpuscular volume (“MCV”) as microcytic, normocytic, and macrocytic.

FIG. 16B is another schematic of a paper-based assay illustratingmeasurements of transport distance in a single grade of paper forquantifying the MCV.

FIG. 17 illustrates paper-based devices for accurately placing MCVs intomicrocytic and normocytic bins based on transport distances.

FIG. 18A is a schematic showing screening material strategy based onpore size.

FIG. 18B shows scans of two-layer devices demonstrating thatfluorescently stained CEM-CD4+T lymphocytes are largely retained by atest layer of chromatography paper (11 micrometers (“μm”) pores) butnearly completely transported through a Nylon mesh (70 μm pores) onto ablot pad.

FIG. 19 is a table showing types of cells, their sources, and examplesof surface markers to develop paper-based cytometers based on positiveselection.

FIG. 20A illustrates a schematic of assay in which captured by surfacemarker-specific antibodies immobilized on paper and detected using avariety of colorimetric indicators.

FIG. 20B is a table detailing several methods for detecting capturedcells based on metabolism or antibody conjugated to a detector.

FIG. 20C illustrates preliminary data showing detection of CD4+Tlymphocytes in paper cytometers at clinically high and low cell countsusing either anti-CD4-HRP (with TMB substrate) or WST-8 as reporters.

FIG. 21A illustrates devices that enable the sequential lysis of RBCs,labeling of WBCs, and negative and positive selection of desiredpopulations by immobilized antibodies.

FIG. 21B illustrates preliminary data using WST-8 to visualize thespecific capture and detection of CD4+T lymphocytes in only zonescontaining anti-CD4.

FIG. 21C illustrates arraying individual cytometers to create a devicecapable of performing multiple measurements simultaneously.

FIG. 21D illustrates disassembled devices to reveal capture zones forspecific cell types.

FIG. 22A illustrates controlled transport of mammalian cells in paperwith WBCs filtered via a 6 μm pore layer while more WBCs are transportedthrough 25 μm layer.

FIG. 22B illustrates transport of WBC improved in paper towels withlarge pore sizes (35 μm).

FIG. 23A is a schematic of a microfluidic device showing a paper-basedcytometer for capturing and enriching yeast that binds a protein (MMP-9)that is impregnated in paper (positive selection array).

FIG. 23B is a plot showing the enriched population in the microfluidicdevice of FIG. 23A.

FIG. 24A shows a first image of a porous Nylon mesh with zero cells usedto generate a calibration curve and to evaluate detection limits.

FIG. 24B shows a second image of the porous Nylon mesh of FIG. 24A with500 cells.

FIG. 24C shows a third image of the porous Nylon mesh of FIG. 24A with1,000 cells.

FIG. 24D shows a fourth image of the porous Nylon mesh of FIG. 24A with2,000 cells.

FIG. 24E shows a fifth image of the porous Nylon mesh of FIG. 24A with3,000 cells.

FIG. 24F shows a sixth image of the porous Nylon mesh of FIG. 24A with4,000 cells.

FIG. 24G shows a first image of selective capture of HER2-positivebreast cancer cells (SK-BR-3) with paper impregnated with HER2-bindingprotein 5F7.

FIG. 24H shows a second image of selective capture of HER2-positivebreast cancer cells (SK-BR-3) with no coating.

FIG. 24I shows a third image of selective capture of HER2-negativebreast cancer cells (MDA-MB-231) with paper impregnated withHER2-binding protein 5F7.

While the invention is susceptible to various modifications andalternative forms, specific embodiments have been shown by way ofexample in the drawings and will be described in detail herein. Itshould be understood, however, that the inventions are not intended tobe limited to the particular forms disclosed. Rather, the aspectsdisclosed herein cover all modifications, equivalents, and alternativesfalling within the spirit and scope of the invention as defined by theappended claims.

DETAILED DESCRIPTION

While aspects of the present disclosure are susceptible of embodiment inmany different forms, there is shown in the drawings and will herein bedescribed in detail some embodiments with the understanding that thepresent disclosure is to be considered as an exemplification of theprinciples of the embodiments and is not intended to limit the broadaspect of the inventions to the embodiments illustrated. For purposes ofthe present detailed description, the singular includes the plural andvice versa (unless specifically disclaimed); the words “and” and “or”shall be both conjunctive and disjunctive; the word “all” means “any andall”; the word “any” means “any and all”; and the word “including” means“including without limitation.”

Paper-based microfluidic devices are useful in the separation andquantification of cells in human blood based on two principles: (i)size-exclusion, in which a paper is selected with an ideal pore size topermit or restrict the flow of cells through a device; and (ii) affinityseparation, in which a paper is biochemically functionalized to capturea specific cell of interest based on its expression of surface markers.The microfluidic devices described below in reference to the drawingsare based on these two principles and bring cell-counting capabilitiesdirectly into hands of users at a point-of-need, with the potential torevolutionize diagnostics in a manner similar to, for example, theintroduction of the home pregnancy test.

The microfluidic devices include paper-based analytical andbioanalytical sensors with features that focus at least in part onseparation, detection, and quantification of cells from complex samples.The manufacturing of the microfluidic devices provides simple, yetfunctional, devices from layers of paper and tape, for example. In otherexamples, instead of or in addition to paper, microfluidic deviceinclude any porous materials configured or selected with the proper poresize. The microfluidic devices (i) are designed to allow theincorporation of a number of fluidic operations into paper-basedanalytical sensors, and (ii) are capable of controlling (i.e.,permitting or impeding) the wicking of cells based on the pore sizes ofthe papers and through modifications of the chemical properties of thepaper fibers through simple chemical functionalization reactions. Afurther benefit of the microfluidic devices is the ability tomanufacture paper-based analytical sensors reproducibly and in largevolumes, e.g., hundreds of millions of devices per year per test.Additional benefits of the microfluidic devices include (i)environmental monitoring of toxic or contaminating species, (ii)separations of complex mixtures of biological and non-biological matter,(iii) the study of interactions between microbes and hosts, and (iv) thedevelopment of diagnostic assays that are designed specifically for usein the developing world and limited-resource settings.

Referring to FIGS. 1A-1C, the use of cells in paper for bioanalyticalassays has thus far been limited to those applications where cells areeither impregnated into paper (e.g., to study the effects of nutrientgradients on the growth of cells and models for tumor formation) orfiltered by the paper (e.g., for the detection of sickled red bloodcells or to perform blood-typing assays). In both examples, paper servesas a medium for the broad exclusion or filtration of cells. One factorthat has limited the development of paper-based assays for the detectionof cells is the choice of material. As illustrated in FIG. 1A, asubstrates selected—typically, Whatman chromatography paper Grade no.1—do not wick mammalian cells due to their small average pore size. Theuse of materials with small pore sizes in paper-based devices ishistorical, rather than an absolute requirement. Hundreds of differentgrades of paper are available commercially and provide differentcombinations of pore sizes (ca. 0.2-200 μm), pore size distributions,thicknesses, surface areas, and overall chemical composition (e.g.,cellulose-based or polymer-based). Cells also span a similarly largerange of sizes, as illustrated in FIG. 1B. It follows, as illustrated inFIG. 1C, that a grade of paper could be selected that permits themigration of cells through a paper microfluidic device based on size.The broad range of available pore sizes facilitates the use of stacks ofpaper as size-exclusion media that selectively separates and isolatescells.

Referring to FIGS. 2A and 2B, a number of papers are characterized fortheir ability to permit or restrict the wicking of erythrocytes (i.e.,red blood cells). Focusing on erythrocytes as a model system offersthree important benefits: (i) they are available as stabilized andrelatively purified suspensions in buffer; (ii) they are available inwhole blood; and (iii) the ability to quantify their concentrationprovides a critical hematological index, the hematocrit. The hematocritis the volume fraction of packed erythrocytes in whole blood—the ratioof packed red blood cell (RBC) volume to the total blood volume.Aberrant hematocrit values are related to diseases that are common inthe developing world (e.g., anemia and dehydration). By way of example,in a healthy adult, the hematocrit is typically in the range of about36%-48% for women and 42%-52% for men. Deviations from these ranges aretypically indicative of diseases, such as anemia, which is indicated inlower percentages in patients, and dehydration, which is indicated athigher percentages in patients. Currently, measurements of thehematocrit require either a hematology analyzer or a centrifuge. Neitherinstrument is readily available in limited-resource settings, but theiranalytical capabilities are greatly needed.

For example, as illustrated in FIG. 2A, the properties of paper are usedto measure the hematocrit using a distance X erythrocytes wickedlaterally within a paper-based microfluidic device 100. The microfluidicdevice 100 includes a first (top) layer of paper 102 with aliquid-receiving area 104 through which a liquid sample 106 is receivedinto the microfluidic device 100. The first layer of paper 102 has afirst average pore size that functions as a filter and, for example,restricts flow of white cells.

The microfluidic device 100 further includes a second (bottom) layer ofpaper 108 that is stacked below the first layer of paper 102. The secondlayer of paper 108 has pores of a second average pore size and includesa channel 110 with a thickness T. The channel 110 has a starting end 112positioned at least in part in an overlapping manner with theliquid-receiving area 104 of the first layer of paper 102. The channel110 has a terminating end 114 that extends laterally at a predeterminedwicking distance XT from the starting end 112. The dimensions of thechannel 110, including the wicking distance XT and the channelthickness, are selected based on one or more paper characteristics, suchas average paper pore size, paper pore size distribution, the paperporosity, paper bubble point, and/or paper flow rate. According to oneexample, each layer 102, 108 is a cellulose-based paper, such asAhlstrom chromatography paper. Optionally, the second layer of paper 108is pretreated with ethylenediaminetetraacetic acid (EDTA)and/or sodiumchloride (NaCl) to promote lateral flow of RBCs through the paper matrixformed by the microfluidic device 100.

As illustrated in FIG. 2B, the first average pore size of the firstlayer of paper 102 and the second average pore size of the second layerof paper 108 cause a wicking effect in which at least some of the liquidsample 106 flows along the channel 110 at least a portion of the wickingdistance X between the starting end 112 and the terminating end 114. Forexample, the liquid sample 106 travels a first wicking distance X1 ifthe hematocrit percentage is approximately 55%, a second wickingdistance X2 if the hematocrit percentage is approximately 60%, a thirdwicking distance X3 if the hematocrit percentage is approximately 45%,and a fourth wicking distance X4 if the hematocrit percentage isapproximately 35%. Accordingly, the average pore size of the layers ofpaper 102, 108 is selected such that the liquid sample 106 fills thechannel 110 a shorter wicking distance X if (a) the liquid sample 106has a higher concentration of the cells of interest (e.g., RBCs) than if(b) the liquid sample 106 has a lower concentration of the cells ofinterest.

The channel 110 has a thicker initial area, which extends approximatelythe same distance as the second wicking distance X2, and a narrowersecondary area that extends to the end of the total wicking distance XT.The thicker initial area is much thicker than the general thickness T ofthe channel 110 in the secondary area (as illustrated in FIG. 2A). Thethicker initial area permits the wicking of higher hematocrits(e.g., >60%) laterally by having a wider (or thicker) initial channelarea. The initial wide area provides sufficient area for a highconcentration of cells to migrate through the channel 110 with minimalaggregation, while not wicking beyond the initial wide area. However,the thinner secondary area of thickness T is provided to obtain resolvedwicking distances for lower hematocrits (e.g., 30%-55%). According toone example, the initial wide area is approximately 5 millimeters thickby 10 millimeters long, and the thinner secondary area of thickness T isapproximately 2 millimeters thick by 40 millimeters long.

The hematocrit percentage is optionally indicated via one or more flowindicators 116 that indicate certain percentage marks (e.g., 60, 50, 40,and 30), in a thermometer-styled readout. According to the embodiment ofFIGS. 2A and 2B, the flow indicators 116 are positioned on the secondlayer of paper 108. However, in other embodiments, the flow indicators116 are positioned on the first layer of paper 102, or are positioned onboth layers of paper 102, 108. Additionally, in other embodiments theflow indicators 116 are configured to indicate any cell concentration ofa liquid sample (not just hematocrit percentages).

The layers of paper 102, 108 are stacked in direct contact with eachother or are stacked in close proximity with each other. For example,the second layer of paper 108 is optionally separated from the firstlayer of paper 102 by an air gap to facilitate ease of wicking flow ofthe liquid sample 106. In another example, an intermediate layer isinterposed between the layers of paper 102, 108 to facilitate theattachment of the layers of paper 102, 108 to each other. For example,the intermediate layer is a removable adhesive that facilitates thetemporary affixing of the layers 102, 108 for ease of assembly and/ordisassembly of the microfluidic device 100.

This exemplary approach relies on the obstruction of pores within theplane of the paper, which occurs in proportion to the concentration oferythrocytes that is applied to the device. That is, the pores within alayer of paper are easier to obstruct with relatively large numbers oferythrocytes than with a lower concentration of cells. As a result of abottlenecking effect, high hematocrits stop migrating sooner than lowhematocrits, and the inherent red color of the erythrocytes provides alabel-free indication of signal transduction. The wicking distances Xmeasured in paper-based devices correlate to hematocrits, which areoptionally measured using standard techniques, and the wicking distanceX that RBCs wick laterally is proportional to the hematocrit as measuredin the flow channel 110. According to one test, the sample test duration(or incubation period) is approximately 30 minutes.

In accordance with the illustrated embodiment, the first layer of paper102 extends only near the liquid-receiving area 104. However, in otherembodiments, the first layer of paper 102 extends away from theliquid-receiving area 104. For example, in one alternative embodiment,the first layer of paper 102 is similar or identical in length to thesecond layer of paper 108.

In accordance with another alternative embodiment, the microfluidicdevice 100 includes a laminate layer 120 affixed to a top surface of thechannel 110 to prevent or minimize flow of liquid away from the areanear the channel 110, such as flow towards the first layer of paper 102.Additionally or alternatively, a laminate layer 122 is affixed to abottom surface of the second layer of paper 108.

Referring to FIG. 3A, the effect of paper pore size on the performanceof a paper-based hematocrit assay illustrates that small pore sizesrestrict the transport of RBCs within a device, while larger pore sizesallow the RBC to migrate freely. Specifically, and by way of exampleonly, a first microfluidic device 200 with a first average pore size ofabout 6 micrometers (μm) restrict the flow of RBCs to a first wickingdistance Y1, a second microfluidic device 202 with a second average poresize of about 10 micrometers (μm) restrict the flow of RBCs to a secondwicking distance Y2, a third microfluidic device 204 with a thirdaverage pore size of about 15 micrometers (μm) restrict the flow of RBCsto a third wicking distance Y3, and a fourth microfluidic device 206with a fourth average pore size of about 25 micrometers (μm) restrictthe flow of RBCs to a fourth wicking distance Y4. As the average poresize increases, so does the respective wicking distance.

Referring to FIG. 3B, in an alternative embodiment and by way of afurther example, the third average pore size of about 15 micrometers(μm) is a threshold pore size that designates a maximum limit beyondwhich the performance level remains approximately the same for the samehematocrit assay. Accordingly, in this alternative embodiment theperformance remains the same for both the third average pore size ofabout 15 micrometers (μm) and for the fourth average pore size of about25 micrometers (μm). In other words, the third and fourth wickingdistances Y3 and Y4 are approximately the same.

Referring to FIG. 4A, separations of cells from complex media suggestthe ability to fractionate populations of leukocytes contained in thebuffy coat of blood into its different components (e.g., neutrophils andlymphocytes). The capability to, for example, separate neutrophils andlymphocytes provides an important hematological index related toleukocyte counts that is useful in assessing susceptibility toinfections or undesirable side-effects caused by drug treatments. Thus,based on a size-exclusion feature in which differences in cell size is adeterminative factor, a microfluidic device 300 has paper layers withpore sizes selected to separate large granulocytes 302 from smalllymphocytes 304. The microfluidic device 300 prevents the largegranulocytes 302 from passing through the microfluidic device 300, whilethe small lymphocytes 304 are permitted to pass through the microfluidicdevice 300.

Referring to FIG. 4B, the use of microfluidic paper-based devices isalso expected to address the important topic of livestock health, whichis related to food quality, by performing analytical assessments of thequality of milk from cows and goats. In such separations—related tospoilage or mastitis—the background components of milk (i.e., colloidsof casein and milk fat globules) are highly heterogeneous and present aunique matrix from which contaminating cells (i.e., somatic cells andbacteria) are separated. For example, a microfluidic device 310 haspaper layers with pore sizes to separate somatic cells 312 from bacteria314.

Referring to FIG. 5, a microfluidic device 400 include a first layer ofpaper 402, with a polymer material 403 grafted onto the paper, and asecond layer of paper 404. Erythrocytes 406—the major component of wholeblood—are expected to present a significant source of interference forseparations in which whole blood is the sample. If fractionation basedon size is insufficient, additional and/or alternatives approaches areused to improve the quality of the separations. For example, inreference to differences in adhesive properties that exist betweensurfaces of erythrocytes 406 and leukocytes 408, an inexpensive polymerof glucose 403 (e.g., dextran) is to cause the aggregation oferythrocytes. Erythrocytes 406—and not leukocytes 408—partitionpreferentially into dextran-rich aqueous phases. Accordingly, based onthis feature, the dextran material 403 that is grafted onto the firstlayer of paper 402 as a vehicle via which contaminating erythrocytes 406are specifically removed from paper-based separation media.

The polymer material 403 can be any adhesive material that causes cellsof a first type (e.g., erythrocytes 406) from a liquid sample to bind tothe first layer of paper 402. Additionally, the average pore size of thefirst layer of paper 402 causes cells of a second type (e.g., leukocytes408) to flow through a first liquid-receiving area 410 of themicrofluidic device to a second liquid-receiving area 412. The secondliquid-receiving area 412 is positioned at least in part in anoverlapping manner with the first liquid-receiving area 410. The averagepore size of the second layer of paper 404 causes the cells of thesecond type to wick through the second layer of paper 404.

In another exemplary approach, simple centrifugation methods (e.g., anegg-beater centrifuge) are used to stratify cells by density prior tointroducing the fraction containing the least dense cells—rich inleukocytes—into paper-based devices. In yet another exemplary approach,a reagent (e.g., saponin) causes the selective hemolysis oferythrocytes. This approach likely requires devices to undergo rigorouswashing to remove contaminants (e.g., hemoglobin) that are released bylysis.

Referring to FIG. 6A, a comprehensive fractionation of cells from wholeblood is achieved with a 3D microfluidic device 500 having multiplelayers of stacked paper. The 3D microfluidic device 500 separatesmultiple biological species from a single liquid sample 502 based on theseveral layers of paper 504-508, with each layer being a different gradeof paper with different pore sizes. Specifically, a first layer of paper504 has an average pore size of about 20 micrometers (μall), a secondlayer of paper 505 has an average pore size of about 15 micrometers(μm), a third layer of paper 506 has an average pore size of about 6micrometers (μm), a fourth layer of paper 507 has an average pore sizeof about 0.2 micrometers (μm), and a fifth layer of paper 508 includes ablot that does not include any cells, except potentially very smallbacteria.

The microfluidic device 500 is assembled and the liquid sample 502 isadded to a liquid-receiving area 510. The layers of paper 504-508 arestacked in an overlapping manner with respect to each other. Then, themicrofluidic device 500 is disassembled and the layers of paper 504-508are isolated to examine the separated cells. After disassembly, and byway of example, the first layer of paper 504 has retained only largewhite blood cells 512, the second layer of paper 505 has retained onlysmall white blood cells 513, the third layer of paper 506 has retainedonly red blood cells 514, the fourth layer of paper 507 has retainedonly bacteria 515, and the fifth layer of paper 508 has retained plasma516. In other words, each layer of paper 504-508 has an average poresize that is smaller than an average diameter of the respective cells ofinterest form the liquid sample 502. For example, the first layer ofpaper 504 has an average pore size—of about 20 micrometers (m)—that issmaller than an average diameter of the large white blood cells 512.

Specific cell types and purified plasma is separated and stored withinthe layers of the 3D microfluidic device, which enables downstreamand/or off-site analysis of samples of blood. In addition to using theseseparations in limited-resource settings, similar paper-based 3Dmicrofluidic devices also enable: (i) purification of viruses from cellculture supernatants, and (ii) subsequent culture of microorganisms thathave been separated using paper-based devices.

According to the illustrated embodiment, the average pore size decreasesfrom the top layer 504 to the bottom layer 508. Based on specificapplications, in accordance with other embodiments, the average poresize is the same in at least two of the plurality of layers 504-508and/or the average pore size increases from the top layer 504 to thebottom layer 508.

Referring to FIG. 6B, a 3D microfluidic device 600 has multiple layersof stacked papers for separating multiple biological species from asingle liquid sample 602, based on several layers of paper 604-608. Ablot 610 collects any remaining cells and/or fragments from the sample602. The microfluidic device 600 is generally similar to themicrofluidic device 500 illustrated in FIG. 6A, except that one or moreof the paper layers 604-608 include a plurality of adhesive areastreated with the same or different adhesive materials.

By way of example, one or more of the paper layers 604-608 is treatedwith a cocktail of antibodies to separate and detect cells from thesample 602. For example, a second paper layer 605 is treated with acocktail of antibodies including anti-CD71 and anti-CD47, and a thirdpaper layer 606 is treated with a single type of antibodies—anti-CD34.The second and third paper layers 605, 606 are treated in a single area(similar to the layers of the microfluidic device 500). In contrast,each of a fourth layer 607 and a fifth layer 608 is treated withantibodies in multiple areas 1-4. For example, the fourth layer 607 istreated with anti-CD15 in area 1, anti-CD177 in area 2, anti-CD193 inarea 3, and anti-siglec8 in area 4. The fifth layer 608 is treated withanti-CD3 in area 1, anti-CD14 in area 2, anti-CD16 in area 3, andanti-CD19 in area 4.

According to one example, in response to adding the sample 602 to theassembled layers of the microfluidic device 600, cells are separated anddetected as follows. Reticulocytes and erythrocytes are separated anddetected in the second layer 605, rare stem cells are recovered in thethird layer 606, granulocytes are recovered in area 1 of the fourthlayer 607, neutrophils are recovered in area 2 of the fourth layer 607,basophils are recovered in area 3 of the fourth layer 607, eosinophilsare recovered in area 4 of the fourth layer 607, T lymphocytes arerecovered in area 1 of the fifth layer 608, monocytes are recovered inarea 2 of the fifth layer 608, NK cells are recovered in area 3 of thefifth layer 608, B lymphocytes are recovered in area 4 of the fifthlayer 608, and other cells and/or fragments are recovered in the blot610.

Referring to FIG. 7, the graphical representation shows varioushematocrit percentages for samples of whole blood using microfluidicdevices treated with sodium chloride and ethylenediaminetetraacetic acid(EDTA). The solid line is a linear fit of the data series (R²=0.985).Each data point is the average of five replicates and the error bars arestandard error of the mean.

Referring to FIG. 8, the graphical representation shows varioushematocrit percentages for samples of whole blood using microfluidicdevices treated with EDTA on two different sections of the microfluidicdevices (i.e., top layer or bottom layer). The solid line is the linearfit of the data series. Each data point is the average of threereplicates and the error bars are standard error of the mean.

Referring to FIG. 9, the graphical representation shows varioushematocrit percentages for samples of isolated red blood cells inAlsever's solution. The solid line is a linear fit of the data series(R²=0.987). Each data point is the average of five replicates and theerror bars are standard error of the mean.

Referring to FIG. 10, the graphical representation shows varioushematocrit percentages for samples of whole blood using microfluidicdevices treated with sodium chloride with and without EDTA. The EDTA wastreated on a lateral channel of the microfluidic device. Each data pointfor the EDTA-treated microfluidic-device data series is the average ofthree replicates and the error bars are standard error of the mean. Eachdata point for the data series of the microfluidic devices without EDTAtreatment is the average of five replicates and the error bars arestandard error of the mean.

Referring to FIG. 11, the graphical representation compares differencesin performance between isolated RBCs and whole blood. Every data pointfor each data series is the average of five replicates and the errorbars are standard error of the mean.

Referring to FIG. 12, the graphical representation shows a comparison ofperformance of isolated RBCs on microfluidic devices that are treatedand that are not treated with sodium chloride. Every data point for eachdata series is the average of five replicates and the error bars arestandard error of the mean.

Referring generally to FIGS. 13A-13C, an alternative embodimentillustrates a microfluidic device 700 for simultaneously determiningboth a hematocrit amount and a hemoglobin amount from a liquid (e.g.,blood) sample 706. Independently, hematocrit and hemoglobin measurementsprovide clinicians with important information about a patient's healthstatus. The microfluidic device 700 is beneficial at least because thesetwo values can be provided simultaneously at the bedside or at apoint-of-care. The assay implemented by the microfluidic device 700facilitates the use of paper in multi-step, autonomous analyses ofblood. Accordingly, the microfluidic device 700 provides one example ofsimultaneous detection of multiple hematological indices and othersoluble markers of health (e.g., creatinine and glucose) usinginexpensive and disposable paper-based devices.

Referring specifically to FIG. 13A, the microfluidic device 700 includesa sample-addition layer 702 with a liquid-receiving area 704 throughwhich the blood sample 706 is received into the microfluidic device 700.The sample-addition layer 702 is made from a porous material (e.g.,paper) having a selected average pore size as described above inreference, for example, to the first layer of paper 102.

The microfluidic device 700 further includes a readout layer 708 that isstacked below, but not adjacent to, the sample-addition layer 702. Thereadout layer 708 is made from a porous material (e.g., paper) having aselected average pore size, for example, as described above in referenceto the second layer of paper 108. The readout layer 708 includes a firstchannel 710 with a thickness T that has a starting end 712 positioned atleast in part in an overlapping manner with the liquid-receiving area704. The first channel 710 further has a terminating end 714 thatextends laterally at a predetermined wicking distance XT from thestarting end 712 in an X direction.

The readout layer 708 further has a second channel 711 that is adjacentto the first channel 710 and that is intended to receive a differenttype of cells from the liquid sample 706 than the type of cells receivedin the first channel 710. For example, the microfluidic device 700 isconfigured such that the first channel 710 indicates a hematocritpercentage in the liquid sample 706 and the second channel 711 indicatesthe hemoglobin level in the liquid sample 706. Although the secondchannel 711 is illustrated in FIG. 13A as a generally circular area, itsshape and size can vary based on specific requirements. For example,according to an alternative embodiment, the second channel 711 issimilar or identical in size and/or shape to the first channel 710.

The microfluidic device 700 further includes a sample-splitting layer705 that is located between the sample-addition layer 702 and thereadout layer 708. The sample-splitting layer 705 includes a firstaperture area 705A through which at least some of the liquid sample 706flows towards the first channel 710, and a second aperture area 705Bthrough which at least some of the liquid sample 706 flows towards thesecond channel 711. According to an alternative embodiment, the apertureareas 705A, 705B are configured in the form of channels.

The microfluidic device 700 also includes a plasma separation membrane707 that is located between the sample-splitting layer 705 and thereadout layer 708. The membrane 707, according to this example, isconfigured with a size and shape that extends only between the secondaperture area 705B of the sample-splitting layer 705 (which is above)and the second channel 711 of the readout layer 708 (which is below). Inother words, the membrane 707 does not extend or act as a barrierbetween the first aperture area 705A of the sample-splitting layer 705and the first channel 710 of the readout layer 708.

As such, according to one specific example, the membrane 707 filterscell debris from plasma remaining in the second aperture area 705B, toallow hemoglobin to be detected by a colorimetric reaction. Optionally,the pores of each of the sample-addition layer 702, the readout layer708, the sample-splitting layer 705, and/or the membrane 707 areselected in accordance with the description provided above in referenceto one or more of FIGS. 1A-12 (e.g., to cause a wicking effect throughone or more of the first channel 710, the second channel 711, the firstaperture 705A, and/or the second aperture 705B).

Referring specifically to FIGS. 13B and 13C, the microfluidic device 700indicates a visual measurement based on the liquid sample 706. Forexample, a blood sample 706 is added to the microfluidic device 700 inthe liquid-receiving area 704 where the blood sample 706 is allowed tosaturate the sample-addition layer 702. The received blood sample 706 issplit uniformly into the two aperture areas 705A, 705B. In the secondaperture area 705B, a reagent (e.g., saponin) is stored to lyse all redblood cells. The plasma separation membrane 707 filters cell debris fromthe remaining plasma to allow hemoglobin to be detected by acolorimetric reaction (e.g., Drabkin's reagent stored on themicrofluidic device 700).

The color formed by the colorimetric reaction is proportional to anamount of hemoglobin (“Hb”). For example, the reaction proceeds from ared color (indicative of low Hb) to a blue color (indicative of highHb). As such, the reaction provides a visual readout of the level of Hbfor a user.

The first aperture area 705A of the sample-splitting layer 705 issaturated with a fixed volume of blood, which allows the hematocrit(“Hct”) assay to proceed in the first channel 710 of the readout layer708. As previously discussed, the Hct assay requires the addition of aknown volume of blood.

Referring to FIGS. 14A-14C, experimental data demonstrates that theamount detected Hb correlates with the measured hematocrit Hct.Specifically, samples of blood with red blood cells (“RBC”) containingmore cells at a higher Hct value have more hemoglobin Hb. For example,FIG. 14A shows that a relatively high 60% Hct value (as indicated by thetraveled distance X1 in the first channel 710) correlates to arelatively high Hb amount (as indicated by a “blue” color in the secondchannel 711). FIG. 14B shows that a relatively medium 45% Hct value (asindicated by the traveled distance X2 in the first channel 710)correlates to a relatively medium Hb amount (as indicated by a changedcolor in the second channel 711). FIG. 14C shows that a relatively low20% Hct value (as indicated by the traveled distance X3 in the firstchannel 710) correlates to a relatively low Hb amount (as indicated by a“red” color in the second channel 711).

According to alternative features of the microfluidic device describedabove, other markers for a multiplexed blood assay include one or moreanalytes of a blood metabolite panel. For example, the analytes includeat least one of glucose, total protein, alkaline phosphatase,creatinine, and blood urea nitrogen (BUN).

Referring to FIGS. 15A-15D, an exemplary embodiment of a multilayermicrofluidic device is configured in accordance with principlesdescribed above. The multilayer microfluidic device has a paper-basedhematocrit assay that is used to illustrate preliminary data incontrolling transport of blood cells to measure Hct. The relationshipbetween blood transport distance and hematocrit range is illustrated inFIG. 15B using an original device design with 50 microliters μL ofblood. Data of FIG. 15C shows the correlation between hematocritpercentage and normalized distance for original device designs using 50μL of blood, as illustrated in FIG. 15B, and scaled device designs usingonly 10 μL of blood, as illustrated in FIG. 15D. Although this examplerefers to paper, in other examples (using in addition or instead ofthose described above and below) include porous materials, e.g., meshes,membranes, or any other materials with pores and solid structures thatdefine pores.

Referring to FIGS. 16A and 16B, schematics show differential transportdistances across multiple grades of paper that are used to bin MCVs asmicrocytic, normocytic, and macrocytic, and to measure transportdistance in a single grade of paper that can be used to quantify theMCV. Papers with large average pore sizes, e.g., >25 μm, should notrestrict the transport of RBCs of any size. Papers of intermediate poresize distinguish RBCs based on their MCV: (i) microcytic cells aretransported farther than normocytic cells in papers with small poresizes; and (ii) transport of macrocytic cells is reduced in comparisonto normocytic cells (FIG. 16A). Additionally, a grade of paper isidentified where transport distance is accurately related to the MCV(FIG. 16B).

Referring to FIG. 17, preliminary data of paper-based devices isintended to obtain accurate placement of MCVs into microcytic andnormocytic bins based on transport distances. Specifically, thepaper-based devices include five blinded samples of whole blood that aresuccessfully differentiated and categorized based on transport distancesin devices prepared from only a single grade of paper. A larger numberof samples is desired to assess the accuracy of correlating transportdistances to MCV.

Referring to FIGS. 18A, 18B, and 19, previous observations fromdeveloping an Hct assay are applied to identify materials that permitWBC transport. To identify porous materials that meet the requiredcriteria for every cell type of interest (e.g., larger neutrophils vs.smaller lymphocytes), the screen extends far beyond traditionalchromatography papers and includes paper towels (e.g., Bounty Basic),synthetic wipers (e.g., TechniCloth), and porous meshes (e.g., forscreen printing). The focus is on materials having pore sizes range from10-100 μm and have porosities that range from 10-80%. The chemicalproperties of the material are also considered: leukocytes arenegatively charged and a positively charged polymer mesh (e.g., Nylon)may lead to undesirable electrostatic capture. Chemical modificationcould provide control over such effects. These efforts parallelporometry and microCT analyses. A combination of material propertiesthat, when matched to the size of each cell type, is expected tomaximize the cell's cross-section and increase its probability ofspecific binding while minimizing non-specific capture by filtration.

The screen of materials using cultures of cells and primary cellsisolated from whole blood is conducted as represented in FIG. 19. Asimple two-layer device is prepared having a test material (to assessfiltration) and a blot pad (to capture all transported cells). Cells arelabeled with a general stain (e.g., Hoechst 33258 or DiO), which enablethe visualization and quantitation of cells on each layer when imaged bymicroscopy, fluorescence imager, or scanner. Preliminary datademonstrates this experimental process comparing Whatman 1chromatography paper (11 μm pore size, substantial filtration) and aNylon mesh (70 μm pore size, negligible filtration) using CEM-CD4+Tlymphocytes with diameters of approximately 11 μm (FIG. 18B).

Referring to FIGS. 20A-20C, a panel of reporters is conjugated tomarker-specific antibodies in a strategy to create a paper-basedcytometer. Options include enzymes (e.g., HRP), metabolic indicators(e.g., WST-8), colloidal particles (e.g., gold), and polymerphotoinitiators. The resulting architecture is analogous to a sandwichimmunoassay—the analyte of interest (i.e., a cell) is captured by anantibody immobilized on a solid support (e.g., paper) and detected usinga paired antibody conjugated to a reporter. Based on the high copynumbers of surface markers, paired antibodies may recognize eitheridentical or unique markers. Reporters are screened with dot-blot styledassays: (i) cells are labeled with antibody conjugates and unboundconjugate are washed away; (ii) labeled cells are spotted onto testzones in single-layer devices to retain all cells and evaluate maximumpossible signal, and (iii) a substrate is added to visualize capturedcells (FIG. 20C). This strategy enables the evaluation of two criticalselection criteria: (i) generation of best visible signals with lowestlimits of detection and greatest dynamic range and (ii) the directcomparison between calibration curves and the observed signals in fullyassembled cytometers, which enable the quantitation of the number ofcaptured cells.

Referring to FIGS. 21A-21D, a paper-based cytometer is designed to havemultiple layers with a single patterned zone. Like all three-dimensionalpaper-based devices, each layer serves a unique function. For example,Layer 1 is for sample addition and potentially stores lysis reagents toprocess blood and eliminate RBCs. Layer 2 stores reporter species usedto label and detect cells. Layer 3 is an optional layer that containsantibodies that bind off-target cells so that only desired cells aretransported (i.e., negative selection). Layer 4 contains antibodies thatbind targeted cells and complete the full sandwich immunocomplexrequired for detection (i.e., positive selection). These antibodies areimmobilized by physisorption or covalent attachment. Layer 5 collectsall unbound cells and waste products. The selection of this materialhelps control assay duration via its wicking rate.

A singleplex cytometer requires only 10 μL of blood to perform ameasurement. Briefly, the assay workflow is as follows: (i) blood isadded to cytometers where RBCs are lysed and WBCs are labeled withreporters (this incubation step helps control assay duration); (ii) washbuffer is added to transport cells through negative and positiveselection zones; (iii) unbound material is collected in the blot (theuse of a wash buffer is commonplace in point-of-care diagnostics, e.g.,OraQuick HIV tests); (iv) the device is delaminated to expose thecapture layer (a peeling step effectively destroys the device, assistswith disposal, and ensures that it is not reused); and (v) substrate isadded, if necessary, to visualize and measure captured cells. Using thisapproach, paper-based cytometers immunophenotype cells for at leastthree markers (e.g., CD45+ reporter/CD20—negative selection/CD14+capture for monocytes). This detection strategy with a cytometerspecifically detects CD4+ T lymphocytes only in channels that containimmobilized anti-CD4 (FIG. 21B). Cell transport is completed in 5minutes and color development using a metabolic indicator requires anadditional 25 minutes.

The analytical performance of paper-based cytometers is evaluated bycreating calibration curves using known cell counts, determining limitsof detection, and determining the dynamic range of the visible response.Assay conditions are tuned to ensure that each cell type is detectableacross its range of clinically-relevant cell counts. Of particularinterest is characterizing the specificity of cytometers for individualcell types when challenged with: (i) high counts of off-target cells(e.g., excess neutrophils for an eosinophil cytometer); (ii) complexmixtures of WBCs prepared from cultures or derived directly from thebuffy coat of whole blood; and (iii) incomplete hemolysis. Afterindividual cytometers are optimized, which includes identification ofnegative or positive selection methods, multiple cytometers are combinedonto a common device (FIG. 21C). Based on previously determined devicescaling rules and the results of singleplex cytometers, a multiplexedcytometer is expected to require approximately 75 μL of blood.

A large panel of clinical blood samples (N>100) is used to compare theperformance of paper-based cytometers to standard hematology analyzers.This population provides insight into the effects that variations in anyindex (e.g., Hct) have on the accuracy of a measurement or diagnosis. Toaddress these concerns, this cohort includes samples from patients withknown presentations of complex conditions detectable by hematological(e.g., microcytic hypochromic anemia) or cytometric (e.g., neutropenia)assessment. Spike-and-recovery assays are used to determine the accuracyof the read guides (FIG. 21D), and the following are determined: (i)sensitivity and specificity, (ii) positive and negative predictivevalues, and (iii) receiver operating characteristic (ROC) curves.

Signals generated by paper-based cytometers are colorimetric and areintended to be interpreted by eye. Ideally, the operator compares thecolor of a test zone a read guide to determine cell counts in theapplied sample (FIG. 21D). To obtain differentials, the operator eitheruses simple math or an additional guide (e.g., bin matching) todetermine the ratio of the counts of each cell type to the total WBCcount (i.e., differential analysis).

Referring to FIGS. 22A and 22B, a controlled transport of mammaliancells in paper is illustrated. For example, materials with larger poresizes or higher porosities transport WBCs and ultimately enable thedevelopment of low-cost devices capable of detecting a brad class ofcells from complex mixtures. According to the specific illustratedexamples, mammalian cells are transported in paper with WBCs filteredvia a 6 μm pore layer while more WBCs are transported through 25 μmlayer (FIG. 22A). Transport of WBC is improved in paper towels withlarge pore sizes, e.g., 35 μm FIG. 22B).

Referring to FIGS. 23A and 23B, enriched yeast in an experiment displaysan antibody that binds matrix metalloproteinase 9 (MMP-9) from asolution where the majority of yeast does not display an MMP-9 bindingprotein. When a mixture of yeast is passed through paper that isimpregnated with recombinant MMP-9 (FIG. 23A, positive selection),capture and specific enrichment of yeast is observed displaying theMMP-9 antibody (FIG. 23B). In contrast, no yeast is observed in paperlacking MMP-9 (FIG. 23A, negative selection). These findings demonstratethe ability to impregnate paper with a protein (MMP-9) and capture cells(yeast) with protein-embedded paper.

Referring to FIGS. 24A-24I, in a proof-of-capability study, HER2-bindingnanobody, 5F7, is spotted onto Nylon capture membranes and physisorptionis relied on as an immobilization strategy. It is determined thatapproximately 25% of the applied 5F7 protein is irreversibly absorbedinto the Nylon mesh. Breast cancer cell capture is evaluated, across arange of concentrations, and 5F7-immobilized materials are created fromporous Nylon to selectively capture HER2-positive SK-BR-3 breast cancercells. Using samples containing cultured SK-BR-3 cells stained with afluorescent dye and suspended in PBS buffer, a calibration curve isgenerated and detection limits are evaluated on porous Nylon meshes(FIGS. 24A-24F). Using fluorescent dyes as the reporter, as few as 500total cells are observed captured by the device (FIG. 24B), although itis proposed that detection is significantly improved using reporterenzymes (e.g., luciferase) to amplify signal. Even so, a limit ofdetection in the range of hundreds to thousands of total cells iscompatible with detecting sub-populations of cells found within a tumorbiopsy. Critically, when cell-permeable Nylon is first impregnated withHER2-binding protein 5F7, HER2-positive SK-BR-3 cells treated with DiO(a commercially available membrane dye, FIG. 24G) are captured. Incontrast, SK-BR-3 cells pass through Nylon that lack 5F7 (FIG. 24H), andfar fewer numbers of HER2-negative MDA-MB-231 cells are captured inNylon containing 5F7 (FIG. 24I).

Each of these embodiments and obvious variations thereof is contemplatedas falling within the spirit and scope of the claimed invention, whichis set forth in the following claims. Moreover, the present conceptsexpressly include any and all combinations and subcombinations of thepreceding elements and aspects.

What is claimed is:
 1. A microfluidic device, comprising: a first layerof a porous material with pores having a first average pore size, thefirst layer having a liquid-receiving area through which a liquid sampleis received into the microfluidic device; a second layer of anotherporous material stacked below the first layer, the second layer havingpores of a second average pore size; and a channel positioned within thesecond layer and having a starting end positioned at least in part in anoverlapping manner with the liquid-receiving area, the channel having aterminating end extending laterally at a predetermined wicking distancefrom the starting end, the first average pore size and the secondaverage pore size causing a wicking effect in which at least some of theliquid sample flows along the channel at least a portion of the wickingdistance between the starting end and the terminating end.
 2. Themicrofluidic device of claim 1, further including flow indicatorsindicative of the wicking distance traveled by the liquid sample alongthe channel.
 3. The d microfluidic device of claim 2, wherein the flowindicators are on the first layer or on the second layer.
 4. Themicrofluidic device of claim 2, wherein the flow indicators areindicative of a cell concentration of the liquid sample.
 5. Themicrofluidic device of claim 1, wherein the second layer is stacked incontact with the first layer.
 6. The microfluidic device of claim 1,wherein the second layer is stacked in close proximity to the firstlayer, the second layer being separated from the first layer by an airgap.
 7. The microfluidic device of claim 1, wherein the first averagepore size and the second average pore size are selected such that theliquid sample fills the channel a shorter wicking distance if (a) theliquid sample has a higher concentration of the cells of interest thanif (b) the liquid sample has a lower concentration of the cells ofinterest.
 8. The microfluidic device of claim 1, wherein both the firstaverage pore size and the second average pore size are the same size. 9.The microfluidic device of claim 1, wherein the first average pore sizeis smaller than the second average pore size.
 10. The microfluidicdevice of claim 1, wherein the first average pore size is larger thanthe second average pore size.
 11. The microfluidic device of claim 1,wherein the first layer extends only near the liquid-receiving area. 12.The microfluidic device of claim 1, wherein the first layer and thesecond layer are removably affixed to each other.
 13. The microfluidicdevice of claim 1, wherein the first layer and the second layer aretemporarily affixed to each other for a sample test duration.
 14. Themicrofluidic device of claim 13, wherein a sample test duration isapproximately 30 minutes.
 15. The microfluidic device of claim 1,wherein dimensions of the channel, including the wicking distance and achannel width, are selected based on one or more characteristics of theporous material or the another porous material including average poresize, pore size distribution, porosity, thickness, bubble point, andflow rate.
 16. The microfluidic device of claim 1, wherein the secondaverage pore size is smaller than an average diameter of cells ofinterest from the liquid sample.
 17. The microfluidic device of claim 1,further comprising one or more additional layers of respective porousmaterials, including a third layer stacked at least in part in anoverlapping manner with and below the second layer, the third layerhaving pores of a third average pore size, the first average pore sizebeing larger than the second average pore size, the second average poresize being larger than the third average pore size.
 18. The microfluidicdevice of claim 1, further comprising a laminate layer affixed to a topsurface of the channel.
 19. The microfluidic device of claim 1, furthercomprising a laminate layer affixed to a bottom surface of the secondlayer.
 20. The microfluidic device of claim 1, wherein at least one ofthe first layer and the second layer is a cellulose-based paper.
 21. Amethod for providing a microfluidic device, the method comprising:providing a first layer of a porous material with pores having a firstaverage pore size, the first layer having a liquid-receiving areathrough which a liquid sample is received into the microfluidic device;stacking below the first layer a second layer of another porous materialhaving pores of a second average pore size; and positioning a channelwithin the second layer, the channel having a starting end positioned atleast in part in an overlapping manner with the liquid-receiving area ofthe first layer, the channel having a terminating end extendinglaterally at a predetermined wicking distance from the starting end;wherein, the first average pore size and the second average pore sizeare selected such that, upon the receiving of the liquid sample, awicking effect is caused in which at least some of the liquid sampleflows along the channel at least a portion of the wicking distancebetween the starting end and the terminating end.
 22. The method ofclaim 21, further comprising providing flow indicators indicative of thewicking distance traveled by the liquid sample along the channel. 23.The method of claim 22, further comprising stacking the second layer incontact with or in close proximity to the first layer.
 24. The method ofclaim 21, further comprising selecting the first average pore size andthe second average pore size such that the liquid sample fills thechannel a shorter wicking distance if (a) the liquid sample has a higherconcentration of the cells of interest than if (b) the liquid sample hasa lower concentration of the cells of interest.
 25. The method of claim21, further comprising affixing a laminate layer to at least one of atop surface of the channel or a bottom surface of the second layer. 26.A paper-based microfluidic device with multiple layers of paper forseparation of cells based on pore size, the microfluidic devicecomprising: a first layer of paper with pores having a first averagepore size and including a first liquid-receiving area through which aliquid sample is received into the microfluidic device, the firstaverage pore size causing cells of a first size from the liquid sampleto wick through the first layer of paper; and a second layer of paperstacked below the first layer of paper and having pores of a secondaverage pore size that is smaller than the first average pore size, thesecond layer of paper including a second liquid-receiving area that ispositioned at least in part in an overlapping manner with the firstliquid-receiving area and through which a remaining portion of theliquid sample is received, the second average pore size causing cells ofa second size from the liquid sample to wick through the second layer ofpaper, the cells of the second size being smaller than the cells of thefirst size.
 27. A paper-based microfluidic device with multiple layersof paper for separation of cells based on cell affinity, themicrofluidic device comprising: a first layer of paper with pores havinga first average pore size and including a first liquid-receiving areathrough which a liquid sample is received into the microfluidic device,the first layer of paper including one or more areas having at least oneadhesive material that causes cells of a first type from the liquidsample to bind to the first layer of paper based on chemical,biochemical, or molecular interactions, the first average pore sizecausing cells of a second type from the liquid sample to flow throughthe first liquid-receiving area; and a second layer of paper stackedbelow the first layer of paper and having pores of a second average poresize, the second layer of paper including a second liquid-receiving areathat is positioned at least in part in an overlapping manner with thefirst liquid-receiving area and through which the cells of the secondtype are received from the first liquid-receiving area, the secondaverage pore size causing the cells of the second type from the liquidsample to wick through the second layer of paper.
 28. A microfluidicdevice, comprising: a sample-addition layer of a first porous materialwith pores having a first average pore size, the sample-addition layerhaving a liquid-receiving area through which a liquid sample is receivedinto the microfluidic device; a sample-splitting layer located adjacentto the sample-addition layer, the sample-splitting layer having a firstaperture for receiving a first portion of the liquid sample and a secondaperture for receiving a second portion of the liquid sample; aseparation membrane located adjacent to the second aperture of thesample-splitting layer, the separation membrane receiving only thesecond portion of the liquid sample from the second aperture; and areadout layer of a second porous material located adjacent theseparation membrane, the second porous material having pores with asecond average pore size that is different than the first average poresize, the readout layer having a first channel configured to receive ina starting end the first portion of the liquid sample from thesample-splitting layer, via the separation membrane, the first channelhaving a terminating end extending laterally at a predetermined wickingdistance from the starting end, the first portion of the liquid sampleflowing at least in part along the wicking distance to indicate a firstvalue of the liquid sample, and a second channel being positionedadjacent to the first channel for receiving the second portion of theliquid sample, the second channel including a stored reagent that reactswith the second portion of the liquid sample to indicate a second valueof the liquid sample.
 29. The microfluidic device of claim 28, whereinthe liquid sample is blood, the first value being indicative of ahematocrit percentage in the blood, the second value being indicative ofa hemoglobin value in the blood.
 30. The microfluidic device of claim28, wherein the first portion and the second portion of the liquidsample are split uniformly between the first aperture and the secondaperture of the sample-splitting layer.
 31. The microfluidic device ofclaim 28, wherein the separation membrane is a third porous materialwith pores having a third average pore size that filters cell debrisfrom the liquid sample.
 32. The microfluidic device of claim 28, whereinat least one of the first porous material and the second porous materialis a paper-based material.
 33. The microfluidic device of claim 28,wherein the first value and the second value of the liquid sample areindicated simultaneously.